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Molecular Genetics of Neural Stem Cells
- Sohyun Ahn, PhD, Head, Unit on Developmental Neurogenetics
- Cheol Lee, PhD, Visiting Fellow
- Hui Wang, PhD, Postdoctoral Intramural Research Training Award Fellow
- Sherry Ralls, BA, Technician Biologist
- Anna Kane, BS, Predoctoral Intramural Research Training Award Fellow
- Garrett Cheung, BS, Postbaccalaureate Intramural Research Training Award Fellow
Neural stem cells (NSCs) in the brain reside in specialized microenvironments called niches and produce new neurons throughout the life of an animal. Our overall goal is to understand how the NSCs are established during development and maintained after birth. Using the mouse as a model system, we study the interaction between NSCs and other cells within the neurogenic niche. Combining the power of genetics with other in vivo manipulation techniques, we are currently investigating the nature of regulatory signals presented by other cells in the niche, signals that dictate the behavior of NSCs. Insights gained from these studies should allow the development of cell type–specific strategies for the recruitment of endogenous NSCs during regeneration or for treatment of disease.
Establishment of the postnatal neurogenic niche
Sonic hedgehog (Shh) promotes proliferation of neural stem cells (NSCs) in the adult brain. However, Shh signaling does not act on NSCs until the late gestational stages, suggesting that proliferation of embryonic NSCs (= radial glia cells) and postnatal NSCs are regulated differently. Furthermore, the postnatal neurogenic niche contains various cell types such as ependymal cells that are also derived from embryonic NSCs around birth. Yet, how the distinct niche cell types are specified remains unclear. To better understand the molecular regulatory mechanism of Shh signaling, we focused on the Gli family of transcription factors (Gli1, Gli2, and Gli3), which are activated or modified in response to Shh activity. In particular, Gli3 is processed into a repressor form (Gli3R) in the absence of Shh signal and acts as the major negative transducer of the pathway. We investigated the role of Gli3 as a repressor in two systems in which Shh activity is lacking: the developing dorsal forebrain and embryonic NSCs. Our findings demonstrate the novel role of Gli3R in regulating neural stem/progenitors in the developing brain and in the postnatal neurogenic niche.
Around the time of birth, embryonic radial glia give rise to postnatal NSCs and ependymal cells, which constitute the neurogenic niche in the subventricular zone (SVZ) of the lateral ventricle in the mouse forebrain. Given that Shh–positive signaling begins around this transition, we addressed the role of the Gli3 repressor, in the absence of Shh signaling, in shaping this postnatal neurogenic niche. Our conditional genetic deletion approaches demonstrated that the Gli3 repressor is critical for specifying postnatal ependymal cells and NSCs. First, the Gli3 repressor is required to suppress gp130/STAT3 signaling at the transcriptional level to regulate the amount of GFAP–expressing glia cells in the SVZ. Next, the Gli3 repressor is required to maintain the appropriate amount of Numb protein via LNX ubiquitin ligase. Loss of Numb led to disruption in cell adhesion between ependymal cells and NSCs, resulting in compromised neurogenic activity of neighboring NSCs. Taken together, we provide a novel mechanism for the establishment of the SVZ niche structure and neurogenesis through an interplay between NSCs and environment.
Gene expression profiling of adult neural stem cells and their niche
The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the SVZ of the lateral ventricle. While several molecules produced by the niche cells are known to regulate adult neurogenesis, no systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been conducted. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate SVZ niche cells, including NSCs, transitamplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and micro-dissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. We also obtained the SMEP of NSCs using cDNA microarray technology. Through a combination of multiple screening approaches, we identified several candidate genes with potential relevance for regulating NSC behavior, thus providing new insights into the nature of neurogenic niche signals.
Sonic hedgehog signaling in midbrain dopaminergic neuron development
The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. The diverse molecular identities, spatial and anatomical distributions, and neural circuitry of mDA neurons determine their functional complexity. We are interested in how these neurons are specified during embryonic development, focusing on Sonic hedgehog signaling and using the mouse as a model system.
Sonic hedgehog (Shh) signaling is critical for various developmental processes including specification of mDA neurons in the ventral mesencephalon (vMes). While the timing of Shh and its response gene Gli1 segregates mDA neurons, the genes' overall lineage contribution to mDA neurons heavily overlaps (i.e., cells derived from Shh or Gli1 expression are largely the same population). We demonstrated that the same set of mDA neuron progenitors sequentially respond to Shh signaling (Gli1 expression), induce Shh expression, and then turn off Shh responsiveness. Thus, at any given developmental stage, cells rarely coexpress Shh and Gli1. Using ShhCre:GFP mice to delete the Smoothened receptor in the Shh pathway, we demonstrated that loss of Shh signaling in Shh–expressing cells results in a transient increase in proliferation and subsequent depletion of mDA neuron progenitors in the posterior vMes, as a result of facilitated cell cycle exit. Moreover, the change in duration of Shh signaling in vMes progenitors altered the timing of their contribution to the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc) mDA neurons. Taken together, our investigation into the relationship between Shh–secreting and –responding cells revealed an intricate regulation of induction and cessation of Shh signaling, which influences the distribution of mDA neurons in the VTA and SNc.
- Hayes L, Ralls S, Wang H, Ahn S. Duration of Shh signaling contributes to mDA neuron diversity. Dev Biol 2013;374:115-126.
- Lee C, Hu J, Ralls S, Kitamura T, Loh YP, Yang Y, Mukouyama Y, Ahn S. The molecular profiles of neural stem cell niche in the adult subventricular zone. PLoS One 2012;7:e50501.
- Wang H, Ge G, Uchida Y, Luu B, Ahn S. Gli3 is required for maintenance and fate specification of cortical progenitors. J Neurosci 2011;31:6440-6448.
- Hayes L, Zhang Z, Albert P, Zervas M, Ahn S. The timing of Sonic hedgehog and Gli1 expression segregates midbrain dopamine neurons. J Comp Neurol 2011;519:3001-3018.
- Regina Armstrong, PhD, Uniformed Services University of the Health Service, Bethesda, MD
- Hwai-Jong Cheng, MD, PhD, University of California, Davis, CA
- Michael J. Holtzman, MD, Washington University, St. Louis, MO
- Y.P. Loh, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
- Yosuke Mukoyama, PhD, Genetics and Developmental Biology Center, NHLBI, Bethesda, MD
- Mark Zervas, PhD, Brown University, Providence, RI